Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

Mechanistic insights into cancer cell killing through interaction of phosphodiesterase 3A and schlafen family member 12.

Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.

Rogaratinib: A potent and selective pan-FGFR inhibitor with broad antitumor activity in FGFR-overexpressing preclinical cancer models.

Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756).

Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

Overexpression of the urokinase receptor splice variant uPAR-del4/5 in breast cancer cells affects cell adhesion and invasion in a dose-dependent manner and modulates transcription of tumor-associated genes.

mRNA levels of the urokinase receptor splice variant uPAR-del4/5 are associated with prognosis in breast cancer. Its overexpression in cancer cells affects tumor biologically relevant processes. In the present study, individual breast cancer cell clones displaying low vs. high uPAR-del4/5 expression were analyzed demonstrating that uPAR-del4/5 leads to reduced cell adhesion and invasion in a dose-dependent manner. Additionally,matrix metalloproteinase-9 (MMP-9) was found to be strongly upregulated in uPAR-del4/5 overexpressing compared to vector control cells. uPAR-del4/5 may thus play an important role in the regulation of the extracellular proteolytic network and, by this, influence the metastatic potential of breast cancer cells.

A Small Molecule-Drug Conjugate (SMDC) Consisting of a Modified Camptothecin Payload Linked to an αVß3 Binder for the Treatment of Multiple Cancer Types.

To improve tumor selectivity of cytotoxic agents, we designed VIP236, a small molecule-drug conjugate consisting of an αVß3 integrin binder linked to a modified camptothecin payload (VIP126), which is released by the enzyme neutrophil elastase (NE) in the tumor microenvironment (TME). The tumor targeting and pharmacokinetics of VIP236 were studied in tumor-bearing mice by in vivo near-infrared imaging and by analyzing tumor and plasma samples. The efficacy of VIP236 was investigated in a panel of cancer cell lines in vitro, and in MX-1, NCI-H69, and SW480 murine xenograft models. Imaging studies with the αVß3 binder demonstrated efficient tumor targeting. Administration of VIP126 via VIP236 resulted in a 10-fold improvement in the tumor/plasma ratio of VIP126 compared with VIP126 administered alone. Unlike SN38, VIP126 is not a substrate of P-gp and BCRP drug transporters. VIP236 presented strong cytotoxic activity in the presence of NE. VIP236 treatment resulted in tumor regressions and very good tolerability in all in vivo models tested. VIP236 represents a novel approach for delivering a potent cytotoxic agent by utilizing αVß3 as a targeting moiety and NE in the TME to release the VIP126 payload-designed for high permeability and low efflux-directly into the tumor stroma.

Overexpression of the urokinase receptor mRNA splice variant uPAR-del4/5 affects tumor-associated processes of breast cancer cells in vitro and in vivo.

uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through Matrigel, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.

Tissue inhibitor of metalloproteinases-1-induced scattered liver metastasis is mediated by host-derived urokinase-type plasminogen activator.

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP-1-induced modulation of a pro-metastatic microenvironment in the liver. Indeed, in livers of uPA-ablated mice elevated TIMP-1 levels did not trigger HGF signalling and did not promote metastasis of a murine T-lymphoma cell line. In contrast, lack of tumour cell-derived uPA induced by gene silencing did not interfere with this pro-metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP-1 levels. This newly identified co-operation between TIMP-1 and host uPA suggests that therapies, simultaneously interfering with pro- and anti-proteolytic pathways may be beneficial for patients with metastatic disease.

Distinct functionality of tumor cell-derived gelatinases during formation of liver metastases.

The specific spatiotemporal role of the matrix metalloproteinase 2 (MMP-2) and MMP-9 (gelatinase) during metastasis is still under debate. Host cells have been described as major contributors to these MMPs during metastasis. Here, we show strong overexpression of MMP-2 and MMP-9 by tumor cells of clinical liver specimen of recurrent metachronous metastases, leading us to address the importance of tumor cell-derived MMP-2 or MMP-9 during liver metastasis. Thus far, distinction of their roles was impossible due to lack of inhibitors which can act exclusively on tumor cells or distinguish MMP-2 from MMP-9. We therefore used short hairpin RNA interference technology in the well-established syngeneic L-CI.5s lymphoma model, in which we could analyze the time course of experimental liver colonization (arrest/invasion of single tumor cells, outgrowth, and invasion within the parenchyma) in immunocompetent mice and correlate these steps with MMP-2 or MMP-9 expression levels. In parental tumor cells, MMP-9 expression closely correlated with the invasive phases of liver colonization, whereas MMP-2 expression remained unaltered. Specific knockdown of MMP-9 revealed a close correlation between invasion-dependent events and tumor cell-derived MMP-9 expression. In contrast, knockdown of MMP-2 did not significantly alter the metastatic potential of the cells but led to a marked inhibition of metastatic foci growth. These findings explain the efficacy of gelatinase-specific synthetic inhibitors on invasion and growth of tumor cells and attribute distinct functions of MMP-2 and MMP-9 to aspects of liver metastasis.

Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling.

Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon.

Optimization of PDE3A Modulators for SLFN12-Dependent Cancer Cell Killing.

6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, potently and selectively inhibits phosphodiesterases 3A and 3B (PDE3A and PDE3B) and kills cancer cells by inducing PDE3A/B interactions with SFLN12. The structure-activity relationship (SAR) of DNMDP analogs was evaluated using a phenotypic viability assay, resulting in several compounds with suitable pharmacokinetic properties for in vivo analysis. One of these compounds, BRD9500, was active in an SK-MEL-3 xenograft model of cancer.

Preclinical Efficacy of the Novel Monocarboxylate Transporter 1 Inhibitor BAY-8002 and Associated Markers of Resistance.

The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor-resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285-96. ©2018 AACR.

Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host.

Tumor cell invasion and metastasis are associated with degradation of components of the extracellular matrix by different proteinases. Among those, papain-like cysteine proteases, such as cathepsin B, seem to play an important role, as they are associated with poor clinical outcome in different cancers. In this study, we tested whether cystatin C, a natural extracellular inhibitor of papain-like cysteine proteases, can inhibit metastasis when overexpressed at the tumor-host interface. Local overexpression of cystatin C in liver and lungs of CD1 nu/nu mice was achieved by gene transfer with a novel adenoviral construct, which also led to the presence of 60 ng/mL of cystatin C in the serum. Three days after gene transfer, these mice were challenged by i.v. inoculation of lacZ-tagged human fibrosarcoma cells (HT1080lacZ-K15), leading to the formation of experimental lung and liver metastases. In this model, formation of experimental metastatic foci correlated with expression of cathepsin B in lungs, whereas there was no correlation with metastasis to the liver. In mice overexpressing cystatin C, the number of lung metastases was significantly reduced by 92%, as compared with mice receiving control adenovirus. The efficacy of extravasation of HT1080lacZ-K15 cells into the liver was not affected, indicating the independence of this process from the activity of cysteine-cathepsins. The present report is the first evidence of successful reduction of metastasis by inhibition of cysteine-cathepsins by cystatin C overexpression in the host microenvironment. Furthermore, organ-specific protease expression during tumor-host cell interactions could affect the success of antiproteolytic intervention against metastasis.

Antimetastatic activity of a novel mechanism-based gelatinase inhibitor.

Matrix metalloproteinases (MMPs), and in particular gelatinases (MMP-2 and MMP-9), play a key role in cancer progression. However, clinical trials in which MMP inhibitors were tested in cancer patients have been disappointing. Whereas many reasons have been postulated to explain the failure of the clinical trials, lack of inhibitor selectivity was a major limitation. Thus, despite the consensus opinion that MMP-mediated proteolysis is essential for cancer progression and that certain MMPs represent important targets for intervention, effective and selective inhibition of those MMPs remains a major challenge in drug development. We previously reported the first mechanism-based MMP inhibitor, designated SB-3CT, which is a selective gelatinase inhibitor. Here we report that SB-3CT (5-50 mg/kg/d) is a potent inhibitor of liver metastasis and increases survival in an aggressive mouse model of T-cell lymphoma. This study shows that mechanism-based inhibition of gelatinases represents a novel approach to inhibitor design that promises to be a successful anticancer therapy.

Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells.

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.

Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Increase in gelatinase-specificity of matrix metalloproteinase inhibitors correlates with antimetastatic efficacy in a T-cell lymphoma model.

The recognition that matrix metalloproteinases (MMPs) facilitate tumor cell invasion and metastasis has led to the development of synthetic MMP inhibitors (MMPIs) as cancer therapeutic agents. Because several Phase III trials failed recently to show efficacy of broad-spectrum MMPIs in advanced cancer, the feasibility of MMPs as therapeutic targets has been challenged. However, it has not yet been determined whether MMPIs that have increased specificity may have greater benefit. We show that MMP-9 expression closely correlates with the progression of liver metastasis in a T-cell lymphoma model. MMPIs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data demonstrate a link between increased specificity of MMPIs and enhanced anticancer activity.

Preclinical Efficacy of the Auristatin-Based Antibody-Drug Conjugate BAY 1187982 for the Treatment of FGFR2-Positive Solid Tumors.

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody-drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line-based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331-9. ©2016 AACR.

Knockdown of platinum-induced growth differentiation factor 15 abrogates p27-mediated tumor growth delay in the chemoresistant ovarian cancer model A2780cis.

Molecular mechanisms underlying the development of resistance to platinum-based treatment in patients with ovarian cancer remain poorly understood. This is mainly due to the lack of appropriate in vivo models allowing the identification of resistance-related factors. In this study, we used human whole-genome microarrays and linear model analysis to identify potential resistance-related genes by comparing the expression profiles of the parental human ovarian cancer model A2780 and its platinum-resistant variant A2780cis before and after carboplatin treatment in vivo. Growth differentiation factor 15 (GDF15) was identified as one of five potential resistance-related genes in the A2780cis tumor model. Although A2780-bearing mice showed a strong carboplatin-induced increase of GDF15 plasma levels, the basal higher GDF15 plasma levels of A2780cis-bearing mice showed no further increase after short-term or long-term carboplatin treatment. This correlated with a decreased DNA damage response, enhanced AKT survival signaling and abrogated cell cycle arrest in the carboplatin-treated A2780cis tumors. Furthermore, knockdown of GDF15 in A2780cis cells did not alter cell proliferation but enhanced cell migration and colony size in vitro. Interestingly, in vivo knockdown of GDF15 in the A2780cis model led to a basal-enhanced tumor growth, but increased sensitivity to carboplatin treatment as compared to the control-transduced A2780cis tumors. This was associated with larger necrotic areas, a lobular tumor structure and increased p53 and p16 expression of the carboplatin-treated shGDF15-A2780cis tumors. Furthermore, shRNA-mediated GDF15 knockdown abrogated p27 expression as compared to control-transduced A2780cis tumors. In conclusion, these data show that GDF15 may contribute to carboplatin resistance by suppressing tumor growth through p27. These data show that GDF15 might serve as a novel treatment target in women with platinum-resistant ovarian cancer.